1. Field of the Invention
The present invention relates to an immunoassay for quantitatively determining a trace constituent present in a sample, in which a reaction between an antigen and an antibody is utilized. More particularly, the present invention relates to a homogenous enzyme immunoassay for quantitatively determining an analyte antigen (ligand) in the sample by the use of an enzyme-labeled antibody.
2. Related Art
Analyses of the constituents originated from the living body or chemicals contained in the body fluids, such as blood or urine, are useful for diagnosing the condition of diseases or judging the course of curing, and thus they occupy important parts in the field of clinical test. The so-called enzyme immunoassay has been known in the art as one method for analyzing such constituents (ligands) generally present in a small amount in the body fluids. The enzyme immunoassay may be classified into a heterogeneous system for which B/F (Bound/Free) separation must be effected, and a homogeneous system in which B/F (Bound/Free) separation is not necessary. Meantime, B stands for the labeling material in a complex formed by binding of the specific antibody (or specific antigen) to the ligand, and F stands for the free labeling material which is not bound to the ligand.
In the heterogeneous system, the antigen-antibody bound (B) formed by the reaction between the antigen and antibody is separated from free antibody and antigen (F) by any suitable means and then the activity of the labeling enzyme in the antigen-antibody bound is determined. Although it is expected that the heterogeneous system has a high sensitivity in principle since the bound (B) is separated from free antibody and antigen (F), there is a problem that cumbersome operations are needed for the B/F separation and thus a relatively long time is necessary for the determination.
On the other hand, the reactions in the homogeneous system are based on the phenomenon that the enzymatic activity of the labeling enzyme is affected by some interference caused by binding of an antibody to the antigen (ligand), and the inhibition due to antigen-antibody binding is generally utilized. In general, the antigen is labeled with an enzyme so that the suppression in enzymatic activity either by a steric hindrance imposed on binding of the enzyme, which is bound to a generally large molecule antibody, with the substrate or by a change in three-dimensional structure of the enzyme is detected. For example, EMIT (Enzyme Multiplied Immunoassay Technique) is well known as such a system.
Alternatively, when the antigen is a high molecular weight substance, the antibody may be labeled with an enzyme and the suppression in enzymatic activity due to the antigen-antibody binding reaction may be utilized. The operations in the homogeneous system are thus relatively simplified since complicated B/F separation is not necessary. However, the homogeneous system has a disadvantage that the sensitivity thereof is lower in principle than that of the heterogeneous system.
Improved homegeneous immunoassaying processes for heightening the sensitivity have been disclosed in Unexamined Japanese Patent Publications (KOKAI) Nos. 108756/1985 (corresponding to U.S. Pat. No. 4,692,404), 295466/1991 (corresponding U.S. Pat. No. 5,569,598 and EP 0451848) and 128655/1992 (corresponding U.S. Ser. No. 08/946,685). In these prior-proposed processes, a water-insoluble high molecular weight substrate is used as the substrate for the enzyme. The enzymatic reaction takes place on the surface of the substrate particles which are giant molecules. As a result, the suppression in enzymatic activity due to steric hindrance caused by binding between the enzyme-labeled antibody and the antigen is exaggerated.
In the process disclosed in Unexamined Japanese Patent Publication No. 108756/1985 (U.S. Pat. No. 4,692,404), an antibody is subjected to a competitive reaction with an antigen (ligand) and an enzyme-labeled antigen, and the enzymatic activity of the labeling enzyme after the reaction is detected using an insoluble high molecular weight substrate.
The process described in Unexamined Japanese Patent Publication No. 295466/1991 (corresponding to U.S. Pat. No. 5,569,598) is for the assay of a high molecular weight antigen, in which the high molecular weight antigen (analyte) is reacted with an enzyme-labeled antibody. The enzyme of the enzyme-labeled antibody, which is bound to the high molecular weight antigen, cannot exhibit enzymatic activity on a water-insoluble substrate by steric hindrance. Accordingly, the quantity of enzymatic reaction products decreases with an increase in the quantity of the antigen to be analyzed.
The process described in Unexamined Japanese Patent Publication No. 128655/1992 is for the assay of a low molecular weight antigen, in which a polymerized antigen (i.e., a conjugate or linked product of the antigen (ligand) and a high molecular weight compound) is used. A competitive reaction is effected among the polymerized antigen, the antigen (analyte) and the enzyme-labeled antibody. In the enzyme-labeled antibody bound to the polymerized antigen, the enzymatic activity of the labeling enzyme to a water-insoluble substrate is interfered or hindered by steric hindrance. On the contrary, the enzyme of the enzyme-labeled antibody, which is combined with the antigen (ligand) contained in a sample, keeps its original enzymatic activity to the water-insoluble substrate. Accordingly, the quantity of the enzymatic reaction product increases in proportion to the quantity of the antigen in the sample.
In Unexamined Japanese Patent Publications Nos. 295466/1991 and 128655/1992, the sensitivity of the analysis is improved further by carrying out the above-described immunoreaction and enzymatic reaction in the layer construction of a dry immunoassay element. The immunoassay element has a reagent layer containing a fragmenting enzyme for further fragmenting the decomposition product produced by the labeling enzyme, so that the fragmented lower molecular weight product is detected for further sensitization of the element.
In the above-described manners, the quantities of high molecular weight and low molecular weight antigens (analytes) can be analyzed with a good sensitivity in accordance with the immunoassays described in Unexamined Japanese Patent Publications (KOKAI) Nos. 295466/1991 and 128655/1992, respectively.
When the molecular weight of the antigen falls within an intermediate range between those described in No. 295466/1991 and No. 128655/1992 of Unexamined Japanese Patent Publication, however, a sufficient sensitivity is sometimes unavailable by either of the above-described immunoassays.
In Unexamined Japanese Patent Publication No. 295466/1991, the difference in the enzymatic activity between an antigen-antibody-enzyme complex and an antibody-enzyme complex is detected. The antigen (analyte) is accordingly required to have such a molecular weight as to reveal an interference or inhibition to the enzymatic activity, when binding to the enzyme-labeled antibody. When an intact IgG molecule (M.W. ca. 160,000) is used as the antibody, the active site of the labeling enzyme has already been affected to some extent by the linkage with this IgG. For increasing the steric hindrance against the enzymatic activity by the binding of the antigen, the antigen (analyte) is required to have a molecular weight sufficient for the molecular weight of the antibody which has already be linked. Even if Fab or Fab' fragment (M.W. ca. 50,000) is employed as the antibody, the antigen (analyte) must have a molecular weight sufficient for it. It is considered to be possible to detect the difference in the enzymatic activity between the antigen-antibody-enzyme complex and the antibody-enzyme complex when the antigen has, for example, a molecular weight of 20,000 daltons or greater, preferably about 50,000 daltons or more. Even if the antigen has a molecular weight falling within the above-described range, however, the detection sensitivity lowers with a decrease in the molecular weight of the antigen.
When the antigen (analyte) has a small molecular weight, on the other hand, a polymerized antigen having an increased molecular weight is employed as described in Unexamined Japanese Patent Publication No. 128655/1992. In this case, detected is a difference in the enzymatic activity between the polymerized antigen-antibody-enzyme complex and the antigen (analyte)-antibody-enzyme complex. With an increase in the molecular weight of the antigen (analyte), the enzymatic activity exhibited by the antigen(analyte)-antibody-enzyme complex is suppressed, leading to a reduction in the difference with the enzymatic activity exhibited by the polymerized antigen-antibody-enzyme complex. This immunoassay system is applicable to the antigen (analyte) having a molecular weight of about 20,000 daltons or less, but the less the molecular weight of the antigen, the better the detection sensitivity. On the contrary, with an increase in the molecular weight of the antigen, the detection sensitivity lowers.
Thus, neither of the above-described immunoassay systems brings about sufficient detection sensitivity when an antigen (analyte) has an intermediate molecular weight, for example, within a range of 10,000 to 70,000.